Research on the pathogenic bacterium Haemophilus influenzae has been hindered by the lack of a genetic map. Our objective is to construct a restriction map of the H. influenzae Rd genome and to localize known mutations and cloned genes on this map. In Preliminary work using pulsed-field agarose gel electrophoresis, we have identified four restriction enzymes, APaI, EagI, NaeI and SmaI, that produce ranges of fragment sizes useful for mapping, and we have shown that the genome is about 1840 kb in size. We will map these fragments by Southern analysis, using radiolabelled fragments from one digest to probe blots of the other digests. Cloned Genes will be similarly mapped to specific restriction fragments. Known selectable mutations and antibiotic resistance markers will be mapped by transformation with gel-purified fragments, allowing the restriction map to be combined with existing genetic linkage data.